Abstract Background To evaluate the utility of glucose chemical exchange saturation transfer (glucoCEST) MRI with non-contrast injection in predicting the histological grade of rectal cancer. Methods This prospective analysis included 60 patients with preoperative rectal cancer who underwent pelvic glucoCEST, amide proton transfer-weighted imaging (APTWI), and diffusion-weighted imaging (DWI). In total, 21 low-grade and 39 high-grade cases were confirmed by postoperative pathology. The MTRasym (1.2 ppm), MTRasym (3.5 ppm), and apparent diffusion coefficient (ADC) values of lesions between the low-grade and high-grade groups were compared. The area under the receiver operating characteristic curve (AUC) was generated to evaluate the diagnostic performance of each technique. Logistic regression (LR) analysis was applied to determine independent predictors and for multi-parameter combined diagnosis. Results Elevated MTRasym (1.2 ppm), MTRasym (3.5 ppm) values and lower ADC values were observed in the high-grade group compared with low-grade cases (all p < 0.01). The AUCs of MTRasym (1.2 ppm), MTRasym (3.5 ppm), and ADC for differentiating between low- and high-grade rectal cancer cases were 0.792, 0.839, and 0.855, respectively. The diagnostic performance of the combination of the three indexes was improved (AUC, 0.969; sensitivity, 95.24%; specificity, 87.18%). The good consistency and reliability of the combination of independent predictors were demonstrated by calibration curve analysis and DCA. Conclusion The glucoCEST MRI without contrast injection, APTWI, and DWI all facilitate the assessment of histological grade in rectal cancer, and the combination of the three can effectively discriminate between high- and low-grade rectal cancer, which is expected to be a promising imaging marker. Critical relevance statement The glucose chemical exchange saturation transfer MRI method facilitates the assessment of histological grade in rectal cancer and offers additional information to improve the diagnostic performance of amide proton transfer-weighted imaging, and diffusion-weighted imaging. Key Points Glucose chemical exchange saturation transfer imaging could differentiate histological grade. Amide proton transfer-weighted and diffusion-weighted were associated with histological grade. The combination of different parameters showed the best diagnostic performance. Graphical Abstract